PROJECT SUMMARY/ABSTRACT Cilia are cell projections built around a bundle of specialized microtubules (the axoneme) that mediate essential motile and sensory functions. Cilia propel cells, generate extracellular fluid flows, support signal transduction and release extracellular vesicles. The length of cilium is an important parameter. Motile cilia that are either too short or too long fail to generate or respond to hydrodynamic forces. In mammals, hedgehog signaling is compromised by mutations that alter the cilium length. Genetic studies led to the discovery of several highly conserved kinases that negatively regulate cilium length including: MAK/ICK, LF4/MOK, LF2/CCRK/DYF-18, CNK/NEK/NRK and LF5/CDKL5. Mutations in these kinases cause diseases (ciliopathies) including developmental disorders (endocrine-cerebro- osteodysplasia syndrome, short rib polydactyly syndrome), retinitis pigmentosa, polycystic kidney disease and juvenile epilepsy. How the activity of cilium length kinases is regulated and how they act to adjust cilium length is poorly understood. The steady-state cilium length is dependent on a balance between the rates of ciliary assembly (driven by intraflagellar transport that delivers precursors) and ciliary disassembly pathway that removes axoneme subunits. The identity of the sensor that measures cilium length is unknown. How the activity of cilium length kinases affects the executive pathways that adjust cilium length (intraflagellar transport and disassembly) is poorly understood. Identification of phosphorylation substrates of cilium length kinases will help in revealing the underlying mechanistic principle of cilium length sensing and adjustment. The cilium length kinases are conserved from ciliated protists to man. We will use a unicellular model with a high density of cilia, Tetrahymena thermophila, an excellent genetic and biochemical model, for a novel genetic suppressor screen, based on a gain of function mutation in a conserved cilium length kinase, LF4/MOK, that causes shortening of cilia and cell paralysis. We will use this screen and a new pipeline for comparative whole genome sequencing, to identify extragenic suppressions of LF4/MOK gain of function. This screen has strong potential for identification of novel LF4 interactors including its phosphorylation substrates, opposing phosphatases and upstream activators. The nature of LF4 interactors will dictate functional studies into the mechanism of sensing and execution of cilium length.